Stimulation of thrombocytopoiesis decreases platelet β2 but not β1 or β3 integrins

The expression of CD29, CD61, CD18 and CD11a on platelets was examined by flow cytometry in mice treated with leukaemia inhibitory factor (LIF) or megakaryocyte growth and development factor (PEG-rHuMGDF or mpl-ligand). Treatment for 7–14 d with PEG-rHuMGDF or LIF increased the number of platelets in peripheral blood from 0.9 up to <2.0×10⁶/μl. These treatments decreased the expression of CD11a and CD18, whereas that of CD29 or CD61 was not markedly changed. Study after various doses or times of PEG-rHuMGDF administration indicated that a decrease of CD18 expression occurred when platelet counts started to rise. Platelet RNA content was increased in mice treated with PEG-rHuMGDF but double staining indicated that expression of CD18 was not correlated with RNA content. To evaluate integrin expression as a function of time in circulation, platelets were biotinylated in vivo . In normal or PEG-rHuMGDF-treated mice, the expression of CD29 or CD61 did not change, whereas that of CD18 decreased significantly as a function of time in circulation. These findings indicate, firstly, that stimulation of thrombocytopoiesis leads to the release of platelets with a low content of β2 integrin and, secondly, that this integrin is also selectively lost while in the circulation.     

β2 glycoprotein-I antigen is increased in primary hyperlipidaemia

Summary. In order to determine whether elevated levels of β₂ glycoprotein-I (β₂GPI) are associated with increased plasma lipids, we measured plasma β₂ GPI antigen levels in 47 patients with primary hyperlipidaemia (20 severe hypercholesterolaemia, nine severe hypertriglyceridaemia, and 18 mixed hyperlipidaemia) and 34 normal healthy subjects. Mean β₂GPI levels were significantly increased in each patient group (302.3, 272.9 and 299.1 mg/1. respectively) compared to controls (199.6 mg/1) (p<0.01). Significant correlations were demonstrated between β₂GPI levels and triglyceride and total cholesterol levels in the control group (r = 0.387, r =0.559: P<0.5), but were not observed in all patient groups. These results indicate that β₂GPI is increased in hyperlipidaemia and that its distribution between plasma lipid fractions is perturbed. Plasma lipid levels should therfore be considered when interpreting results of β₂GPI antigen assays.     

A novel β-thalassaemia mutation in the 5'untranslated region of the β-globin gene

Summary. A thymidine deletion at position +10 of the 5'untranslated region of the β-globin gene was detected in a β-thalassaemia intermedia patient carrying a β; 39 stop codon mutation on the other chromosome; this new mutation, +10(-T), was detected by automated fluorescent DNA sequencing and verified by dot-blot allele-specific hybridizations.
The +10(-T) mutation is a 'silent carrier', is associated with a reduced amount of steady-state β-globin mRNA, and establishes a connection between the 5'untranslated region of the β-globin gene and the regulation of its expression.     

Decreased interleukin-2 receptor β chain expression by peripheral blood lymphocytes in chronic liver disease

To investigate the decrease in natural killer (NK) activity in chronic liver disease, interleukin-2 receptor beta chain (IL-2R beta) expression was assessed by peripheral blood lymphocytes (PBL) using flow cytometry and an IL-2R beta chain-specific mouse monoclonal antibody. The percentage of IL-2R beta chain-positive PBL was significantly decreased in patients with chronic viral hepatitis, liver cirrhosis and hepatocellular carcinoma in comparison with normal controls (P less than 0.01). Among chronic viral hepatitis patients, it was significantly less in those with chronic active hepatitis than in those with chronic persistent hepatitis (P less than 0.05). Two-colour flow cytometry revealed that the IL-2R beta chain was mainly expressed by CD8+ or CD16+ cells in both the controls and the liver disease patients. CD8dull+ cells (NK cells) constituted more than 60% of the CD8+ cells expressing the IL-2R beta chain. Expression of the IL-2R beta chain with CD8 or CD16 was also significantly decreased in chronic liver disease patients compared with controls. In chronic viral hepatitis, there was a significant correlation between NK activity and the percentage of IL-2R beta+ PBL (P less than 0.001, r = 0.916), as well as between NK activity and the percentage of PBL co-expressing both the IL-2R beta chain and CD16 (P less than 0.001, r = 0.850). These findings suggest that decreased expression of the IL-2R beta chain by PBL may result in diminished NK activity in chronic liver disease.     

Anti β2 glycoprotein I antibodies: detection and association with thrombosis

Summary. It has been demonstrated that antiphospholipid antibodies (aPL) recognize epitopes formed by anionic phospholipids and protein cofactors. β₂ glycoprotein I (β₂GPI) is accepted as the cofactor of anticardiolipin antibodies (aCL). In the present study we explored the presence and clinical associations of anti β₂GPI antibodies of IgG isotype (aβ₂GPI-IgG), measured by ELISA. We studied sera from 169 patients with aCL and/or lupus anticoagulant (LA), including 52 patients with systemic lupus erythematosus and 49 with primary antiphospholipid syndrome (PAPS). We found 31.9% positive sera for aβGPI-IgG in the whole population and 48.6% in the aCL-IgG(+) group. There was a good correlation between the titre of aCL-IgG and the optical density for aβGPI-IgG ( r = 0.69, P <0.01). The presence of aβ₂GPI-IgG was associated with the presence of aCL-IgG ( P <0.0001) and LA ( P <0.0005). However, none of 23 LA (+) patients without aCL had aβ₂GPI-IgG.
We found a statistically significant association between the presence of aβ₂GPI-IgG and a history of venous thromboembolism (VTE) in our patients ( P <0.005). This association was observed in PAPS ( P <0.05) but not in secondary antiphospholipid syndrome (SAPS).
Our study confirms that some aPL(+) sera react with β₂GPI in special experimental conditions. In addition, the presence of these antibodies is associated with a history of VTE.     

A novel amber mutation in a β°-thalassaemia gene (β37TGG→TAG), with direct detection by mapping the restriction fragments in amplified genomic DNA

Summary. A novel amber mutation, a G to A substitution at the second position of codon 3 7 in the β-globin gene that changes the tryptophan coding triplet (TGG) to a termination codon (TAG), was found in a Chinese β-thalassaemia carrier. The mutant gene creates an additional Dde I recognition site and eliminates the Ava II site, so this point mutation can be directly identified by restriction enzyme analysis.     

Allopurinol improves scavenging ability of the liver after ischemia/reperfusion injury

Abstract: Deterioration of energy metabolism and oxidative stress represent fundamental mechanisms in ischemia and reperfusion injury. In a normothermic ischemia/reperfusion rat model, we investigated whether allopurinol (ALL) may improve the scavenging ability of the liver after ischemia. ALL was given prior to ischemia and reperfusion (concentration 100 or 50 mg/kg) and controls were given a placebo. After a basal period of 30 min, 1 h normothermic ischemia was induced in the median and left liver lobes followed by 24 h observation. The overall liver function was assessed by bile secretion, and free oxygen production was assessed by glutathione efflux into bile during the first 60 min of reperfusion and at 24 h. Allopurinol (concentration 100 mg/kg) protected hepatocyte function as bile flow improved significantly in this group after 1 and 24 h of reperfusion compared with that of controls. Oxidative stress was also significantly attenuated in this group, as efflux of glutathione into bile was significantly higher in the ALL group (100 mg/kg) after 24 h but not after 1 h of reperfusion compared with that of controls. ALL given in a concentration 50 mg/kg had some, but a non-significant, effect. We conclude that biliary glutathione is an important marker of oxidative stress and may reflect the scavenging ability of the liver after ischemic injury. Significant correlation of bile flow with biliary glutathione during reperfusion indicates that oxidative stress is an important mechanism attenuating liver function after ischemia/reperfusion injury.     

Polyenylphosphatidylcholine pretreatment protects rat liver from ischemia/reperfusion injury

Background

Hepatic injury induced by ischemia/reperfusion following surgery, transplantation, or circulatory shock combined with resuscitation is a major clinical problem. Polyenylphosphatidylcholine (PPC) has strong antioxidant, cytoprotective and anti-inflammatory effects.

Aim

In this study, the influence of PPC pretreatment on ischemia-reperfusion (I/R) injury of the liver was examined in rats.

Methods

The animals were divided into three groups: control (n = 10), I/R (n = 15) and I/R + PPC (n = 15). PPC was given 100 mg/day for 7 days before experiment. Several parameters of hepatic damage, oxidative stress, neutrophil infiltration and nuclear factor kappa beta (NF-κB) expression were measured as well as microscopic examination.

Results

We observed that a significant reduction in AST and ALT values in the PPC treated group when compared with the ischemic group. The increases in hepatic total NO₂ + NO₃ and MDA, and decreases in SOD and GSH levels after reperfusion were partially, but significantly, inhibited by PPC pretreatment. I/R induced increase in hepatic myeloperoxidase content and NF-κB expression were also lowered by PPC pretreatment. Animals pretreated with PPC presented minimal hemorrhage and reduced signs of liver injury.

Conclusion

PPC pretretament provided significant protection againts I/R injury to the liver. This treatment could be therapeutic in liver transplantation and other conditions associated with I/R injury.     

Protective effect of sulfhydryl-containing antioxidants against ischemia/reperfusion injury of prepubertal rat intestine

Background and Aim:  Reactive oxygen species generated during reperfusion of the tissue are known to play an important role in the basic pathophysiology of ischemia/reperfusion (I/R) injury. The aim of this study was to investigate and compare the protective effects of three sulfide-based antioxidants, N -acetylcysteine (NAC), erdosteine (ERD), and α-lipoic acid (LA), on I/R injury of the small intestine tissue.
Methods:  Forty male Sprague–Dawley rats weighing between 100–150 g were divided into five groups ( n  = 8 for each): control (sham operated), I/R, I/R + NAC, I/R + ERD, and I/R + LA. Intestinal ischemia was provided by occluding the superior mesenteric artery via a special microvascular clamp; ischemia for 30 min and reperfusion for 3 days were carried out. Ileal specimens were obtained to determine the tissue levels of malondialdehyde (MDA), protein carbonyl contents (PCO), superoxide dismutase (SOD), and glutathione peroxidase (GPx) activities and histological changes.
Results:  The rats subjected to intestinal I/R exhibited an increase in tissue MDA and PCO; the levels could hardly be ameliorated in the treatment groups. SOD and GPx activities were significantly decreased in the I/R group, whereas their reduction was less expressed in the treatment groups. Additionally, the histopathological injury scores of the disulfide-treated groups were lower than those of the I/R group.
Conclusion:  All of the sulfhydryl-containing antioxidants used in this study exhibited a significant role in attenuating intestinal I/R injury; however, the outcome of the LA-treated group was significantly marked than that of the others.     

大鼠肝脏缺血再灌注损伤c-fos表达及预处理的保护作用

目的探讨预处理（preconditioningPc）对大鼠肝脏缺血再灌注（ischemia／reperfusion,I／R）损伤的影响及其机制。方法制备大鼠肝脏原位I／R损伤的模型,采用免疫组织化学技术结合图像分析方法定量检测原癌基因c-fos表达的情况和肝组织脂质过氧化产物丙二醛（MDA）的变化。结果I／R损伤早期可引起损伤区肝细胞核内原癌基因c－fos的大量表达;PC明显减少了c－fos表达的细胞数量以及减轻肝脏脂质过氧化的程度。结论PC对大鼠肝脏I／R损伤有明显的保护作用,可能的机制之一是抑制肝I／R损伤后原癌基因c-fos的表达和灭活自由基减少脂质过氧化物的生成。     

Severe inclusion body β-thalassaemia with haemolysis in a patient double heterozygous for β°-thalassaemia and quadruplicated α-globin gene arrangement of the anti-4.2 type

We describe a new case of an association of alpha-globin gene quadruplication of the anti-4.2 type with beta(0)-thalassaemia. The patient, a young woman of mixed Brazilian-Portuguese origin, suffered from chronic haemolytic anaemia with splenomegaly. Bone marrow supravital staining with brilliant cresyl blue and electron microscopy studies showed large inclusion bodies in about 3% of erythroblasts. Upon immunofluorescent staining these inclusions reacted with a monoclonal antibody to alpha- but not to beta-globin. Analysis of alpha-globin cluster by Southern blotting showed the presence of pathologic fragments specific for the anti-4.2 alpha-globin gene quadruplication. Alpha/beta mRNA ratio was higher than in cases combining alpha-globin triplication and beta(0)-thalassaemia or in cases of beta(0)-thalassaemia heterozygous state alone (18, 14.7 and 10.1 respectively). Our data confirmed the hypothesis that the clinically detectable haemolysis in this beta(0)-thalassaemic patient was due to an unusually high amount of precipitated alpha-globin in erythroid precursors. This considerable excess of alpha-globin chains was due partly to the beta-globin deficit caused by the presence of the beta(0)-thalassaemic gene, but also to the presence of 6 active alpha-globin genes resulting from alpha-globin gene quadruplication in one chromosome.     

Increased erythropoiesis of β-thalassaemia/Hb E proerythroblasts is mediated by high basal levels of ERK1/2 activation

β - thalassaemia is one of the most common inherited anaemias, arising from a partial or complete loss of β-globin chain synthesis. In severe cases, marked bone marrow erythroid hyperplasia, believed to result from erythropoietin (EPO)-mediated feedback from the anaemic condition is common, however, as yet, no study has investigated EPO-mediated signal transduction in thalassaemic erythroid cells. Using proerythroblasts generated from peripheral blood circulating CD34⁺ haematopoietic progenitor cells, the activation of the mitogen-activated protein kinase/extracellular signal-regulated kinases (MAPK/ERKs) pathway was examined under conditions of steady state growth, cytokine deprivation and post-EPO stimulation. Levels of cellular cyclic adenosine monophosphate (cAMP) and Ca²⁺ were determined as was the degree of erythroid expansion. A significantly higher basal level of phosphorylation of ERK1/2 was observed in β-thalassaemia/Hb E proerythroblasts as compared to normal controls, which was coupled with significantly higher levels of both cAMP and Ca²⁺. Modulation of either cAMP or Ca²⁺ or direct inhibition of MAPK/ERK kinase (MEK) reduced basal levels of ERK1/2 phosphorylation, as well as significantly reducing the level of erythroid expansion. These results suggest that, in contrast to current models, hyper proliferation of β-thalassaemia/Hb E proerythroblasts is an intrinsic process driven by higher basal levels of ERK1/2 phosphorylation resulting from deregulation of levels of cAMP and Ca²⁺.     

Interleukin-1β expression in the pineal gland of the rat

Increasing evidence of the neuroimmunomodulatory role of the pineal gland prompted the present study of pineal gland expression of the immunoregulatory cytokine, interleukin (IL)-1beta. IL-1beta was constitutively expressed in the adult gland, with mRNA levels higher in glands collected during the photophase than in those collected during the scotophase of the light:dark cycle. IL-1beta was up-regulated in pineal cultures, after treatment with either norepinephrine (NE) or interferon (IFN)/lipopolysaccharide (LPS). Although both astrocytes and microglia expressed IL-1beta, important differences were found in the cellular expression of this cytokine under in vivo and in vitro conditions. Increased IL-1beta expression by NE ex vivo and the decline in IL-1 expression at night, when NE levels are elevated, can be explained by immunocytochemical data showing that astrocytes are the predominant cell type expressing this cytokine in vivo, whereas IL-1beta-positive cells are predominantly microglia in pineal explants and dispersed cell cultures. These results are consistent with the hypothesis that cytokines secreted by pineal glia (astrocytes and microglia) may have an important regulatory role in the pineal gland.     

Regulation of β-cell mass and function by the Akt/protein kinase B signalling pathway

The insulin receptor substrate-2/phosphoinositide 3-kinase (PI3K) pathway plays a critical role in the regulation of β-cell mass and function, demonstrated both in vitro and in vivo . The serine threonine kinase Akt is one of the promising downstream molecules of this pathway that has been identified as a potential target to regulate function and induce proliferation and survival of β cells. Here we summarize some of the molecular mechanisms, downstream signalling pathways and critical components involved in the regulation of β-cell mass and function by Akt.     

Type 2 diabetes – a matter of failing β-cell neogenesis? Clues from the GK rat model

Now that reduction in β-cell mass has been clearly established in humans with type 2 diabetes mellitus (T2D), the debate focuses on the possible mechanisms responsible for decreased β-cell number. Appropriate inbred rodent models are essential tools for this purpose. The information available from the Goto-Kakizaki (GK) rat, one of the best characterized animal models of spontaneous T2D, is reviewed in such a perspective. We propose that the defective β-cell mass in the GK model reflects mostly a persistently decreased β-cell neogenesis. The data discussed in this review are consistent with the notion that poor proliferation and/or survival of the endocrine precursor cells during GK foetal life will result in a decreased pool of endocrine precursors in the pancreas, and hence an impaired capacity of β-cell neogenesis (either primary in the foetus or compensatory in the newborn and the adult). As we also demonstrated that β-cell neogenesis can be pharmacologically reactivated in the GK model, our work supports, on a more prospective basis, the concept that facilitation of T2D treatment may be obtained through β-cell mass expansion after stimulation of β-cell regeneration/neogenesis in diabetic patients.     

Severe thalassaemia intermedia caused by interaction of homozygosity for α-globin gene triplication with heterozygosity for β thalassaemia

Summary A 3-year-old child was evaluated for β-thalassaemia intermedia. Molecular characterization including β-globin gene sequence analysis revealed heterozygosity for a single β-thalassaemia mutation, IVSI nt1 (GA). In addition the patient was found to be homozygous for α-globin gene triplication (ααα^anti3,7/ααα^anti3,7). The propositus has a significantly more severe phenotype than has been previously reported with this combination of genetic defects. In contrast, four individuals heterozygous for both triplicated α and for β-thalassaemia had a phenotype of thalassaemia minor, and a fifth had very mild thalassaemia intermedia.     

Cytoprotective effects of melatonin against necrosis and apoptosis induced by ischemia/reperfusion injury in rat liver

Abstract:  Melatonin protects against organ ischemia; this effect has mainly been attributed to the antioxidant properties of the indoleamine. This study examined the cytoprotective properties of melatonin against injury to the liver caused by ischemia/reperfusion (I/R). Rats were subjected to 60 min of ischemia followed by 5 hr of reperfusion. Melatonin (10 mg/kg) or the vehicle was administered intraperitoneally 15 min before ischemia and immediately before reperfusion. The serum aminotransferase activity and lipid peroxidation levels were increased markedly by hepatic I/R, which were suppressed significantly by melatonin. In contrast, the glutathione content, which is an index of the cellular redox state, and mitochondrial glutamate dehydrogenase activity, which is a maker of the mitochondrial membrane integrity, were lower in the I/R rats. These decreases were attenuated by melatonin. The rate of mitochondrial swelling, which reflects the extent of the mitochondrial permeability transition, was higher after 5 hr of reperfusion but was attenuated by melatonin. Melatonin limited the release of cytochrome c into the cytosol and the activation of caspase-3 observed in the I/R rats. The melatonin-treated rats showed markedly fewer apoptotic (TUNEL positive) cells and DNA fragmentation than did the I/R rats. These results suggest that melatonin ameliorates I/R-induced hepatocytes damage by inhibiting the level of oxidative stress and the apoptotic pathway. Consequently, melatonin may provide a new pharmacological intervention strategy for hepatic I/R injuries.     

Evaluation of β-cell mass and function in the Göttingen minipig

Increased knowledge about β-cell mass and function is important for our understanding of the pathophysiology of type 2 diabetes (T2DM). The relationship between the two is difficult to study in humans, whereas animal models allow studies of consequences of, for example, reduction of β-cell mass and induction of obesity and procurement of the pancreas for histological examination. An overview of results obtained in the Göttingen minipig in relation to β-cell function, and mass is provided here. Effects of a primary reduction of β-cell mass have indicated that not all of the defects of pulsatile insulin secretion in human T2DM can be explained by reduced β-cell mass. Furthermore, induction of obesity has shown deterioration of β-cell function and morphological changes in the pancreas. As in humans, obesity leads to an increased β-cell volume in the minipig, and based on the increased number of islets, neogenesis of islets is an important factor in expansion of β-cell mass in this species. Measurement of β-cell function as an estimate of β-cell mass is, at present, the only method possible in humans, and this approach has been validated using lean and obese minipigs with a range of β-cell mass. The effects on β-cell function and mass of obesity of longer duration and/or more pronounced hyperglycaemia remains to be determined, but the models developed so far represent a valuable tool for such investigations.